Essential Requirement for IRF8 and SLC15A4 Implicates Plasmacytoid Dendritic Cells in the Pathogenesis of Lupus

Essential requirement for IRF8 and SLC15A4 implicates plasmacytoid dendritic cells in the pathogenesis of lupus

Roberto Baccalaa,1,2, Rosana Gonzalez-Quintiala,1, Amanda L. Blasiusb,1, Ivo Rimanna, Keiko Ozatoc, Dwight H. Konoa, Bruce Beutlerb,d,2,3, and Argyrios N. Theoﬁlopoulosa,2,3

aDepartment of Immunology and Microbial Science and bDepartment of Genetics, The Scripps Research Institute, La Jolla, CA 92037; cProgram in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and dCenter for Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390

Contributed by Bruce Beutler, December 28, 2012 (sent for review December 21, 2012)

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) IFN-producing cells (22, 23). These cells, known as plasmacytoid are intimately involved in the pathogenesis of lupus. However, it DCs (pDCs), are the most potent producers of type I IFNs, a remains to be determined whether these cells are required in vivo functional characteristic attributed to constitutive expression of for disease development, and whether their contribution is re- TLR7, TLR9, and IRF7 and likely signaling from a unique in- stricted to hyperproduction of type I IFNs. To address these issues, tracellular compartment (24–27). The involvement of pDCs in lu- we created lupus-predisposed mice lacking the IFN regulatory factor pus is further suggested by the reduced frequency of these cells in 8 (IRF8) or carrying a mutation that impairs the peptide/histidine patient blood together with increases in afflicted organs, pre- fl transporter solute carrier family 15, member 4 (SLC15A4). IRF8- sumably caused by the attraction of activated pDCs to in ammatory fl deficient NZB mice, lacking pDCs, showed almost complete absence sites (10). Similar increases have been noted in in ammatory tissues ’ of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibod- of patients with Sjögren s syndrome (28), rheumatoid arthritis (29, ies, along with reduced kidney disease. These effects were observed 30), dermatomyositis (31), and psoriasis (32). despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 Collectively, these results suggest that pDCs, acting through stimuli and intact humoral responses to conventional T-dependent type I IFN hyperproduction, are major pathogenic contributors to lupus. Whether the participation of these cells is obligatory and -independent antigens. Moreover, Slc15a4 mutant C57BL/6- lpr remains to be documented in vivo, however. Here, using con- Fas mice, in which pDCs are present but unable to produce type I genic lupus-predisposed mice lacking pDCs (as well as other DC IFNs in response to endosomal TLR ligands, also showed an absence subsets) owing to IRF8 deficiency, or exhibiting pDC-specific of autoantibodies, reduced lymphadenopathy and splenomegaly, defects in endosomal TLR signaling and type I IFN production and extended survival. Taken together, our results demonstrate that owing to Slc15a4 (feeble) mutation, we provide strong evidence pDCs and the production of type I IFNs by these cells are critical that pDCs are indeed required for disease development, and this contributors to the pathogenesis of lupus-like autoimmunity in effect appears to be mediated by hyperproduction of inflamma- these models. Thus, IRF8 and SLC15A4 may provide important tar- tory cytokines, most likely type I IFNs. gets for therapeutic intervention in human lupus. Results + xtensive evidence suggests that type I IFNs are major patho- Absence of pDCs and CD8α DCs in IRF8-Deficient NZB Mice. Studies Egenic effectors in lupus-associated systemic autoimmunity. A in normal background mice have shown that IRF8, a hemato- well-documented pattern of expression of type I IFN-inducible poietic cell-specific transcription factor, is essential for the de- − − genes occurs in peripheral blood mononuclear cells of patients velopment of pDCs (33–35). Accordingly, assessment of Irf8 / with systemic lupus erythematosus (SLE) (1–3), and reduced dis- NZB mice at a young age (3 mo) showed almost complete ab- – + + + ease is observed in some lupus-predisposed mice that either lack sence of pDCs (CD11clowCD11b B220 Siglec-H PDCA1 )in the common receptor (IFNAR) for these cytokines (4, 5) or have spleen (Fig. 1A), and this correlated with undetectable in vivo been treated with IFNAR-blocking antibody (6). Consequently, production of type I IFNs in response to CpG-DNA (Fig. 1B). + attention has focused on defining the cell subsets and signaling Moreover, CD8α conventional DCs (cDCs) were absent and – – − − processes involved in type I IFN production, the mechanisms by CD4 CD8 cDCs were reduced in Irf8 / mice (Fig. 1C), con- which these mediators accelerate disease, and approaches to in- sistent with the levels of IRF8 expression in these subsets of terfere with these pathogenic events. normal mice (35). Total cDC numbers were unaffected, however, + Early in vitro studies showed that type I IFN production can be likely owing to compensatory increases in CD4 cDCs (Fig. induced in normal blood leukocytes by SLE autoantibodies com- 1C), which do not express IRF8 (35). As noted previously with − − plexed with nucleic acid-containing apoptotic/necrotic cell mate- Irf8 / normal background mice (36, 37), IRF8-deficient NZB + rial, and further work demonstrated that this activity is sensitive mice also showed expansion of marginal zone and CD21lowCD23 to RNase and DNase digestion (7, 8). These results were inte- follicular and transitional B cells, but total B-cell and T-cell num- grated in a more comprehensive scheme following the demon- bers were unchanged (data not shown). stration that type I IFN induction by these complexes is mediated by the engagement of endosomal Toll-like receptors (TLRs) (9– 11). Similarly, antigenic cargo containing nucleic acids was found Author contributions: R.B., K.O., D.H.K., B.B., and A.N.T. designed research; R.B., R.G.-Q., to promote B-cell proliferation in a TLR9- or TLR7-dependent A.L.B., and I.R. performed research; R.B., R.G.-Q., A.L.B., I.R., B.B., and A.N.T. analyzed manner, with this effect enhanced by type I IFN signaling (9, 12, data; and R.B. and A.N.T. wrote the paper. 13). The contribution of nucleic acid-sensing TLRs to systemic The authors declare no conflict of interest. autoimmunity was further corroborated by studies in lupus-pre- 1R.B., R.G.-Q., and A.L.B. contributed equally to this work. disposed mice lacking or overexpressing TLR7 and/or TLR9 (14- 2To whom correspondence may be addressed. E-mail: [email protected], Bruce.Beutler@ 20), and in Unc93b1 (3d) mutant mice in which signaling by UTSouthwestern.edu, or [email protected]. endosomal TLRs is extinguished (21). 3B.B. and A.N.T. contributed equally to this work. The cell population involved in type I IFN production in re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sponse to lupus-related immune complexes corresponds to natural 1073/pnas.1222798110/-/DCSupplemental.

2940–2945 | PNAS | February 19, 2013 | vol. 110 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1222798110 Downloaded by guest on September 30, 2021 Fig. 1. Absence of pDCs and CD8α+ cDCs in young IRF8-deficient NZB mice. Mutant and WT NZB mice (age 3 mo; n = 3–6/group) were analyzed for cellular differences in spleen and in vivo responses to CpG challenge. (A) pDC frequency and numbers. Spleen cells were assessed by flow cytometry using anti-CD11c and anti-PDCA-1 antibodies. Similar results were obtained when cells were stained with antibodies to B220, SiglecH, and CD11b (data not shown). (B) In vivo type I IFN production in response to TLR9 engagement. Serum IFN-α levels in CpG-challenged mice were determined by ELISA. (C) cDC frequency and numbers. + + + – – Spleen cells were analyzed by flow cytometry after gating on CD11c cells to identify CD4 , CD8 , and CD4 CD8 cDC subsets. Numbers within the flow cytometry plots correspond to average frequencies of the indicated subsets. Error bars in graphs indicate SD. Asterisks indicate statistical significance (P < 0.05).

Reduced Autoimmunity in IRF8-Deficient NZB Mice. At age 11 mo, cDC subtypes present in these mice could not compensate for serum levels of polyclonal IgM and IgG, as well as IgG1 and the absence of pDCs in response to this TLR9 stimulus. Fur- − − + IgG2a, were similar in Irf8 / and WT NZB mice, whereas IgG2b thermore, CD11c cDCs derived from GM-CSF–differentiated was slightly increased and IgG3 was decreased in the mutant mice bone marrow (BM) cells showed reduced CD86 up-regulation (Fig. 2A). Strikingly, however, autoantibody production was al- and IL-6 production after in vitro stimulation with TLR7 or most completely suppressed in the IRF8-deficient mice. Thus, IgG TLR9 ligands in the presence or absence of IFN-α (Fig. 4). Thus, − − anti-erythrocyte autoantibodies, which are typically detectable in in addition to pDC deficiency, Irf8 / NZB mice exhibited de- − − 100% of WT mice by age 7–8 mo, were undetectable in Irf8 / fective cDC responses to endosomal TLR stimulation. mice even at 11 mo (Fig. 2B). Moreover, IgG2a anti-chromatin autoantibodies (the dominant subclass in WT NZB mice) and IgG B-Cell Responses to TLR Ligands and Exogenous Antigens Are Not anti-nuclear autoantibodies (ANA) were reduced to background Compromised in IRF8-Deficient NZB Mice. IRF8 is known to affect levels, and IgM anti-chromatin autoantibodies were decreased B-cell differentiation and germinal center formation (40), changes substantially (Fig. 2 C and D). Mirroring these serologic changes, that might contribute to reduced autoimmunity in IRF8-deficient − − kidney disease was significantly ameliorated in Irf8 / mice, as mice. To address this possibility, we assessed in vitro B-cell evidenced by reductions in both glomerulonephritis scores (Fig. responses to TLR or BCR engagement and in vivo antibody 2E) and immune (IgG2a, C3) deposits (Fig. 2F). responses to exogenous antigens. In vitro responses of purified − − Splenomegaly and accumulation of T cells and B cells, ob- Irf8 / B cells to TLR7 and TLR9 ligands in the presence or ab- − − served with aging in WT controls, were also decreased in Irf8 / sence of IFN-α were largely conserved, as indicated by efficient up- NZB mice (Fig. 3 A and B). The reduction in T-cell numbers regulation of CD86 and H2-Kd, as well as production of IL-6 and + + affected both CD4 and CD8 subsets and was accompanied by IL-10 (Fig. S1 A–C). Similarly, activation by anti-IgM and anti- + a decrease in activated CD4 Tcells(Fig.3C and D), whereas CD40 cross-linking was comparable in mutant and WT B cells – – − − the reduction in B cells was primarily in the CD21 CD23 subset (Fig. S1 D and E). Moreover, immunization of WT and Irf8 / (Fig. 3E), which includes the so-called “age-associated” Bcells NZB mice with a thymus-independent antigen (TNP-LPS) or (38, 39). In contrast, the expanded population of marginal zone a thymus-dependent antigen (TNP-KLH) produced anti-hapten − − B cells detected in young Irf8 / mice was retained with aging antibody responses of similar titers and affinities (Fig. S2). Thus, (Fig 3E), whereas the frequency of peritoneal B1 cells was un- despite the numerical changes, B-cell responses to endosomal changed. Thus, IRF8 deficiency in NZB mice was associated TLR ligands and to conventional exogenous antigens were largely with decreased humoral and histological disease manifestations, uncompromised in IRF8-deficient NZB mice, suggesting that re- along with reduced activation/expansion of T cells and B cells. duced autoimmunity likely is not due to B-cell functional defects.

Defective cDC Responses to Endosomal TLR Ligands in IRF8-Deﬁcient Feeble Mutation of Slc15a4 Prevents Autoimmunity in Congenic NZB Mice. Because engagement of endosomal TLRs is thought to C57BL/6-Faslpr Mice. To directly examine how pDCs contribute to lpr be a major mechanism of DC activation in lupus, we also ex- systemic autoimmunity, we developed congenic C57BL/6-Fas amined the response of cDCs to TLR7 and TLR9 ligands. As mice carrying the feeble mutation of Slc15a4. In normal back- − − noted above, Irf8 / NZB mice did not produce detectable ground mice with this mutation, development of both pDCs and IMMUNOLOGY amounts of IFN-α after CpG-DNA injection, indicating that the cDCs is intact, but cytokine production, particularly type I IFNs,

Baccala et al. PNAS | February 19, 2013 | vol. 110 | no. 8 | 2941 Downloaded by guest on September 30, 2021 Fig. 2. Reduced autoimmunity in IRF8-deficient NZB mice. Mutant and WT NZB mice (age 11 mo; n = 5–8/group) were examined for serologic and histological disease characteristics. (A) Polyclonal serum Ig levels. (B) Anti-erythrocyte autoantibodies. Individuals with a geometric mean fluorescence intensity >50 were considered positive for anti-RBC autoantibodies. (C) Anti-chromatin autoantibodies. (D) ANAs, with representative individuals shown. (E) Representative PAS- stained kidney sections and glomerulonephritis scores. (F) Kidney deposits (IgG2a and C3), with representative immunofluorescence images shown. Asterisks indicate statistical significance (P < 0.05).

in response to TLR7 or TLR9 ligands is absent only in pDCs the results with the Slc15a4 mutants infer that the contribution of lpr (27). Accordingly, in Slc15a4 mutant C57BL/6-Fas mice, pDCs these cells is mediated by endosomal TLR-dependent production were present at normal frequencies in spleen (Fig. 5A) and dif- of proinflammatory cytokines, primarily type I IFNs. ferentiated efficiently after Flt3L treatment of BM cells, but did The biological effects of IRF8, also known as IFN consensus not produce type I IFNs in response to TLR7 or TLR9 ligands binding protein, have been investigated extensively (40–42). both in vivo and in vitro (Fig. 5B). In contrast, B cells from Slc15a4 IRF8 is an IFN-inducible transcription factor expressed in lpr + – – mutant C57BL/6-Fas mice showed normal in vitro proliferation pDCs, CD8α cDCs, CD4 CD8α cDCs, macrophages, and B to TLR9 stimulation (Fig. 5C) and efficient humoral responses cells. IRF8 forms heterodimeric complexes with several mo- to T-dependent and T-independent antigens (Fig. S3). Re- lecular partners and binds to various IFN stimulatory response markably, at age 8 mo, these mutants had significantly reduced elements, thereby governing expression of a large set of genes IgM anti-chromatin and an almost complete absence of IgG with broad effects in innate and adaptive immune functions. anti-chromatin autoantibodies and ANA (Fig. 5 D and E). In Nonetheless, a dominant characteristic of mice lacking IRF8 is addition, the mutants had significantly decreased hypergamma- the absence of pDCs. globulinemia (8.4 ± 2.9 mg/mL IgG vs 23.6 ± 14.8 mg/mL in WT We found that the age-associated splenomegaly and expansion controls; P < 0.05) and extended survival (Fig. 5F). Reductions of T and B cells typical of NZB mice were largely abrogated in − − + were also observed in lymphadenopathy and splenomegaly (Fig. Irf8 / congenics, accompanied by significant decreases in CD4 6A), with corresponding numerical decreases in cDCs, T cells T-cell activation, autoantibody production, and kidney disease. + + – – (CD4 and CD8 ), and B cells (CD21 CD23 ), whereas pDCs Interestingly, reductions encompassed not only the classical ANA, and double-negative T cells were reduced, but not significantly but also the dominant antierythrocyte response of this strain. The (Fig. 6 B and C). These findings strongly indicate that pDCs, reductions in antinuclear specificities, particularly in the IgG iso- likely through type I IFN hyperproduction, are major contrib- type, likely are related to the absence of pDCs and minimal, if any, utors to the pathogenesis of systemic autoimmunity. endosomal TLR-dependent induction of type I IFNs by self- nucleic acids and related immune complexes. This is consistent Discussion with previous findings with lupus-predisposed mice deficient in We have used two types of genetic modification in mouse models type I IFN or endosomal TLR signaling, which showed decreases of spontaneous lupus-like disease to define the in vivo role of in these autoantibodies and other disease manifestations (11). pDCs in the pathogenesis of systemic autoimmunity. In the first In terms of the role of pDCs in anti-RBC responses, although instance, we created NZB mice deficient for IRF8, a transcription RBCs lose nuclei and mitochondria and thus lack DNA that factor that regulates the development of pDCs, among other might engage TLR9, these cells contain RNA species that might effects, whereas in the second instance, we created C57BL/6- engage TLR7. Alternatively, the initial trigger for the anti-RBC lpr Fas mice carrying a mutation in SLC15A4, a peptide/histidine response might be provided by uptake of nucleated erythroid transporter critical for endosomal TLR signaling and type I IFN precursors or of expelled nuclei, which are surrounded by an production by pDCs. The evidence with the Irf8 mutants strongly intact plasma membrane displaying phosphatidylserine as an suggests that pDCs are required for lupus development, whereas “eat-me” signal (43–45). Any one of these processes could pro-

2942 | www.pnas.org/cgi/doi/10.1073/pnas.1222798110 Baccala et al. Downloaded by guest on September 30, 2021 Fig. 5. Reduced autoimmunity in Slc15a4 mutant C57BL/6-Faslpr mice. Mu- tant and WT mice were analyzed for pDC development and function at age 4mo(n = 3–5/group), disease manifestations at age 8 mo (n = 7–10/group), and survival (n = 10–13/group). (A) Frequency of pDCs in spleen. Cells were Fig. 3. Reduced T-cell and B-cell expansion in older IRF8-deficient NZB assessed by flow cytometry using anti-CD11c and anti–PDCA-1 antibodies. mice. Mutant and WT NZB mice (age 8–11 mo; n = 3/group) were analyzed Similar results were obtained using antibodies to B220, SiglecH, and CD11b for cellular changes in spleen and peritoneal cavity. (A) Spleen weight and (data not shown). (B) In vivo and in vitro type I IFN production in response to cellularity at 11 mo. (B) T-cell and B-cell numbers in spleen at age 11 mo. endosomal TLR stimulation. Serum IFN-α levels in CpG-challenged mice, and + Cells were analyzed by flow cytometry after gating on TCRβ (T cell) or IFN-α production by BM-differentiated pDCs stimulated with CpG or R848, + + + B220 (B cell) populations. (C)CD4 and CD8 T-cell subsets in spleen at were determined by ELISA. (C) In vitro B-cell proliferation in response to TLR9 + age 11 mo. (D) Frequency of activated CD4 T cells in spleen at age 11 mo. engagement. Purified B cells from mutant and WT mice were stimulated in + −/− Gated CD4 T cells were assessed for expression of CD44. (E) B-cell subsets vitro with CpG. B cells from Tlr9 C57BL/6 mice were used as negative in spleen at age 8 mo. Gated B220+IgM+ cells were analyzed for transi- controls. (D) Anti-chromatin autoantibodies. (E) ANA. (F) Survival. Error bars tional T2 and follicular (T2-FO), marginal zone (MZ), and T1 immature and in graphs indicate SD. Asterisks indicate statistical significance (P < 0.05). age-associated (T1-ABC) B cells. Numbers within flow cytometry plots correspond to average frequencies of the indicated subsets. Error bars in graphs indicate SD. Asterisks indicate statistical significance (P < 0.05). nucleic acids, whereas adjuvanticity of complete (CFA) and in- complete (IFA) Freund’s adjuvants, or alum in response to conventional exogenous antigens is mediated by endosomal TLR- vide both antigenic cargo and nucleic acids for efficient activa- independent pathways (46–48). tion of pDCs and antigen presentation by cDCs. We have attributed the disease-reducing effects of IRF8 deficiency to the absence of pDCs. However, IRF8-deficient mice It is of interest that, in contrast to reductions or even absence + of autoantibodies, responses to conventional T-dependent and also lacked CD8α cDCs and had diminished in vitro responses T-independent antigens were of normal magnitude and quality in to endosomal TLR stimulation in the retained cDC subsets. − − fi Irf8 / NZB mice, as reported for normal background mice with These modi cations also may contribute to disease reduction. α+ B-cell–specific conditional Irf8 deletion (37). A possible expla- CD8 cDCs have several functional properties of potential nation for this finding is that with lupus-associated autoantigens, relevance to autoimmunity, including ingestion of materials from dying cells, antigen cross-presentation, and production of IL-12, adjuvanticity is provided by endosomal TLR engagement by self- a cytokine involved in Th1 differentiation (49). Regardless, however, these cells are unlikely to promote disease, because they do not express TLR7, the engagement of which appears to be a major pathogenic contributor in several lupus-predisposed mouse strains (14, 15, 21), and do not produce significant amounts of type I IFNs in response to ligands or viruses that engage endosomal TLRs or cytosolic sensors for nucleic acids (50–54). Similarly, the partial defects in TLR7 and TLR9 + – – responses by the IRF8-deficient CD4 and CD4 CD8α cDCs are likely insufficient to explain disease reduction, given that these subsets have been reported to produce meager amounts of type I IFNs when stimulated with endosomal TLR ligands, including lupus-associated nucleic acid-containing immune complexes (53). Nonetheless, considering our present findings and previous findings of others demonstrating disease reduction in lupus-prone mice lacking all DC subsets (including pDCs) (55), targeted deletion/inactivation and/or reconstitution of specific cDC subsets is needed to definitively clarify the exact role of cDCs, as opposed to pDCs, in systemic autoimmunity. An additional open question is whether modifications in the B-cell compartment caused by IRF8 deficiency also might contribute to disease reduction, given that ablation of IRF8 has been Fig. 4. Defective in vitro responses by cDCs from IRF8-deficient NZB mice. shown to affect B-cell differentiation and germinal center orga- cDCs were differentiated from BM cells from WT and mutant mice (age 3 mo; nization, as well as to induce expansion of marginal zone and – follicular B cells (40). However, as reported previously (37) and pools of 2 3 mice) in the presence of GM-CSF, then stimulated with endo- fi somal TLR ligands in the presence or absence of IFN-α.(A and B) CD86 up- reproduced here with IRF8-de cient NZB mice, humoral + regulation defined by flow cytometry after gating on CD11c cells. (C) IL-6 responses to exogenous antigens remain unmodified. Further- −/− production determined by ELISA. One representative of two independent more, we have shown that Irf8 B cells mounted normal in vitro experiments is shown. Error bars in graphs indicate SD of samples analyzed in responses to TLR7 and TLR9 ligands, which could be enhanced IMMUNOLOGY triplicate. Asterisks indicate statistical significance (P < 0.05). with IFN-α. Our results suggest that B-cell activation by endo-

Baccala et al. PNAS | February 19, 2013 | vol. 110 | no. 8 | 2943 Downloaded by guest on September 30, 2021 cytokine production in pDCs remains to be defined. Nevertheless, the defect seems to be related not to inefficient uptake of TLR ligands or secretion of type I IFNs, but rather to inefficient signaling by endosomal TLRs, given that intracellular Ifna transcripts and corresponding proteins could not be detected in stimulated mutant pDCs even in the presence of exogenous IFN-α (27). A reported function of SLC15A4 is to transport, in a low pH-dependent manner, free histidine from the endosome to the cytosol (60, 61), a process that might facilitate the cathepsin-mediated cleavage of endosomal TLRs required for efficient signaling (62– 65). Alternatively, SLC15A4 may be localized in a unique pDC- associated lysosome-related organelle, where it contributes to the establishment of an optimal microenvironment required for the assembly of functional TLR signaling complexes and hyperpro- Fig. 6. Cellular changes in Slc15a4 mutant C57BL/6-Faslpr mice. Spleen and LNs = duction of type I IFNs (27, 66). of mutant and WT mice were examined at age 8 mo (n 3/group). (A)Spleen In summary, based on our studies with Irf8-deleted and Slc15a4 and LN organ weights. (B) pDC and cDC numbers in spleen. Flow cytometry was + low + mutant mice, we report strong evidence that pDCs are essential used to identify pDCs (PDCA-1 CD11c ) and cDCs (CD11c ). (C)T-cellnumbers + + – – in spleen. CD4 ,CD8 ,andCD4CD8 (double-negative) T cells were identified for the initiation of abnormal innate immune responses leading by flow cytometry after gating on TCRβ+ cells. (D) B-cell numbers in spleen. to systemic autoimmunity, likely through high production of Spleen cells were examined by flow cytometry as in Fig 3E. Error bars in graphs type I IFNs. These events appear to be instrumental for DC indicate SD. Asterisks indicate statistical significance (P < 0.05). activation, efficient antigen presentation by DCs and B cells, and production of disease-inducing isotype-switched autoantibodies. Similar processes are likely involved in the pathogenesis somal TLRs, although necessary, by itself is not sufficient to of human SLE; interestingly, IRF8 and SLC15A4 have been drive autoimmunity, with type I IFN hyperproduction resulting identified as susceptibility loci in certain populations with this from the concomitant engagement of these TLRs in pDCs, re- condition (67, 68). Our present results further suggest that phar- quired as well. This scheme of endosomal TLR engagement in macologic inhibition of IRF8, and particularly interference with both B cells and pDCs fits our previously outlined model for the the effects of SLC15A4 on pDCs, may be of utility in the induction of systemic autoimmunity (56). treatment of SLE and other inflammatory conditions in which Combined with the foregoing considerations, the data from type I IFNs are major pathogenic effectors. IRF8-deficient mice provide strong support for an indispensable in vivo disease-promoting effect of pDCs. However, IRF8 de- Materials and Methods fi fi ciency prevents us from stating a de nite conclusion as to Mice. NZB mice and C57BL/6-Faslpr mice were obtained from Jackson Labo- whether pDCs contribute to disease solely through hyperpro- ratory or The Scripps Research Institute Animal Facility. C57BL/6 mice de- duction of type I IFNs or other functions, such as antigen pre- ficient for IRF8 (Irf8−/−)orTLR9(Tlr9−/−) or carrying the feeble mutation of the fi −/− sentation (57). The recent identi cation of the mutant strain Slc15a4 gene have been reported (27, 35), and congenic Irf8 NZB and feeble, in which defective expression of the peptide/histidine Slc15a4 mutant C57BL/6-Faslpr mice were generated by standard marker- transporter SLC15A4 leads to absence of endosomal TLR sig- assisted breeding as described previously (4). The WT and Irf8−/− NZB mice + − naling and severely reduced production of proinflammatory used in this study were littermates generated by crossing heterozygous Irf8 / cytokines, including type I IFNs, specifically in pDCs (27), has mice. All mice were housed under specific pathogen-free conditions. The provided an experimental tool to address this question. We experimental protocols were performed in accordance with the National lpr found that lupus-predisposed C57BL/6-Fas mice carrying this Institutes of Health’s Guide for the Care and Use of Laboratory Animals and mutation exhibited striking reductions in disease manifestations approved by The Scripps Research Institute’s Animal Care and Use Committee. and extended survival, despite efficient B-cell responses to both endosomal TLR stimuli and conventional exogenous antigens. Statistical Analysis. Group comparisons were analyzed using the unpaired These findings support the critical role of type I IFN produc- two-tailed Student t test. Survival was analyzed by Kaplan–Meier plots and tion by pDCs in systemic autoimmunity. Disease reduction in the log-rank test. P < 0.05 was considered significant. lpr Slc15a4 mutant C57BL/6-Fas mice,aswellasinUnc93b1 mutant lpr C57BL/6-Fas mice (21), is at variance with the reported disease Additional Methods. Procedures for in vivo and in vitro studies, cell prepa- lpr enhancement in IFNAR-deficient MRL-Fas mice (58). There rations, flow cytometry, analysis of ANA and anti-erythrocyte autoantibodies, is no clear explanation for this variance, but possibilities include kidney pathology, and immunohistology are described in SI Materials and genetic contributions beyond the Fas defect, exemplified by differ- Methods. ences in disease severity between these strains (59), and complete absence of signaling in Ifnar1-deleted mice versus pDC-specific ACKNOWLEDGMENTS. We thank Carrie N. Arnold for assistance with the absence of type I IFN production in Slc15a4 mutant mice. B cell proliferation assays and Anthony Nguyen for excellent technical lpr Creation of MRL-Fas mice congenic for the Slc15a4 mutation support. This work was supported by National Institutes of Health Grants Unc93b1 AR53228, AR31203, AR39555, 1U19-AI100627-01, and 2P01-AI070167- (and possibly the mutation) will likely clarify this issue. 06A1. A.L.B. was supported by The Irvington Institute Fellowship Program The exact mechanism by which SLC15A4 deficiency specifically of the Cancer Research Institute. This is article number 22092 from The Scripps compromises endosomal TLR signaling and proinflammatory Research Institute, Department of Immunology and Microbial Sciences.

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